The present invention relates to virus quantitation. More specifically, the invention relates to quantitation of viruses by measuring scattered light.
Determination of virus concentration is important in, among other things, quantitation of viral vectors for use in gene therapy, quantitation of oncolytic viruses and quantitation of virus-based vaccine compositions. The quantitation of viruses for use in gene therapy with accuracy and precision is critical to ensure adequate comparability of data obtained in various intra-and inter-institutional studies, as well as to ensure comparability between virus preparations used for preclinical and clinical studies (Mittereder et al., J. Virol. 70:7498-7509, 1996). The quantitation of viruses that lyse tumor cells (oncolytic viruses) is also important in determining the correct dosage. In addition, quantitation of virus-based vaccines is important for safety and efficacy of administration of these compositions.
Examples of oncolytic viruses include mutated adenovirus (Heise et al., Nat. Med. 3:639-645, 1997), mutated vaccinia virus (Gnant et al., Cancer Res. 59:3396-3403, 1999) and mutated reovirus (Coffey et al., Science 282:1332-1334, 1998). Examples of viral vectors for use in gene therapy include mutated vaccinia virus (Lattime et al., Semin. Oncol. 23:88-100, 1996), mutated herpes simplex virus (Toda et al., Hum. Gene Ther. 9:2177-2185, 1998), mutated adenovirus (U.S. Pat. No. 5,698,443) and mutated retroviruses (Anderson, Nature 392(Suppl.):25-30, 1998).
As early as the 1960s and 1970s, light scattering measurements were used to study the assembly and aggregation of viral components and viral particles (Smith et al., Biochemistry 6:2457-2465, 1967; Cummins et al., Biophys. J. 9:518-546, 1969; Camerini-Otero et al., Biochemistry 13:960-970, 1974). Diffusion coefficients, molecular weights and particle dimensions of viruses and viral components have all been studied with light scattering techniques. These studies have emphasized the variation of light scattering per virus particle, depending on the state of aggregation, association, dissociation, etc., of the virus particles. Modern light scattering detectors are designed to permit characterization of the size distributions of molecules and particles, including viruses, using an auxiliary detector (e.g., ultraviolet light absorbance detector or refractive index detector) as the concentration detector.
U.S. Pat. No. 5,837,520 to Shabram et al. discloses and claims a method for determining the number of intact virus particles in a sample by monitoring the ultraviolet absorbance of the effluent from a column of an anion exchange resin and comparing that absorbance to a standard curve that is prepared with virus suspensions of known concentrations. This method, with measurement of light absorbance at 260 nm and 280 nm, is used by Shabram et al. (Hum. Gene Ther. 8:453-465, 1997) to quantitate adenovirus in suspensions.
Publications on light scattering in the context of virus quantitation emphasize the interference by light scattering with ultraviolet light absorbance measurements (Maizel et al., Virology 36:115-125, 1968; Tikhonenko et al., Mol. Biol. (Moscow) 12:393-395, 1978; Mittereder et al. supra). Tsoka et al. (Biotechnol. Bioeng. 63:290-297, 1999) use dynamic light scattering to detect distributions of particle sizes in suspensions of virus-like particles, and teach that it is necessary to add antibodies to a suspension of virus-like particles in order to induce a change in particle size that can then be measured by dynamic light scattering.
Dynamic light scattering measurements are distinct from static light scattering measurements. Typically, dynamic light scattering (also known as photon correlation spectroscopy) is an optical method used to study the Brownian motion of particles in solution. Measurements are taken to detect fluctuations in the intensity of light scattered by a sample, at time points on a scale related to the time taken for a particle to diffuse a distance comparable to the wavelength of the light scattered. See Tsoka et al. In contrast, static light scattering is not based on fluctuations in intensity over time, and is not directed to detecting Brownian motion or diffusion rates of particles.
Bistocchi et al (Tumori 63:525-534, 1977) describe quantitation of murine mammary tumor virus (muMTV) in mouse milk, and refer to their virus quantitation as having been done by xe2x80x9clight scattering.xe2x80x9d However, this reference does not actually describe the use of a light scattering detector for quantitating viruses, but instead describes the measurement of ultraviolet light absorbance at 260 nm, which is also referred to by those authors as optical density. It is the increase in optical density (i.e. the decrease in transmitted light) that these authors refer to as xe2x80x9clight scattering.xe2x80x9d Light absorbance and light scattering are distinct phenomena: the optical density values of the samples of Bistocchi et al. include contributions from light scattering by the virus particles (which decreases the amount of light transmitted through the sample), as well as contributions from light absorbance at 260 nm by milk proteins and by viral nucleic acids. The authors took into account the expected light absorbance by milk proteins, based on the protein content of the milk as determined by the method of Lowry et al. (J. Biol. Chem. 193:265-275, 1951). However, they apparently assumed that after correcting for the light absorbance due to milk proteins, the result would be a measure of light scattering by virus particles. They did not take into account the light absorbance at 260 nm due to viral nucleic acids. In any case, this reference does not actually report quantitation of a virus by light scattering, but instead reports quantitation of a virus by an adjusted or corrected ultraviolet light absorbance measurement.
An important difference between an absorbance measurement and a light scattering measurement is that the detector for measuring absorbance must be placed on the side of the sample opposite to the light source, along the axis of illumination, where it measures the decrease in light transmitted through the sample, as done by Bistocchi et al. On the other hand, a detector for measuring scattered light is placed away from the axis of illumination, for example at 90 degrees to that axis, to measure the increase in light that is scattered by the sample at a non-zero angle to the incident beam. A second difference between absorbance and scattering measurements stems from the necessity to employ a wavelength that is specifically absorbed by the sample in the former method (e.g. ultraviolet light with a wavelength of 260 or 280 nm), whereas wavelengths that are not absorbed by the sample are preferred in the latter method (e.g. visible light with a wavelength of 690 nm from a diode laser, 632.8 nm from a helium-neon laser or 488 nm from an argon-ion laser).
There is an ongoing need for more accurate methods for measuring virus concentrations, particularly for adenovirus, for which no widely accepted standard method is known. The present invention addresses this need.
The present invention provides a method for determining the number or concentration of virus particles in a sample, including the steps of: measuring the amount of light scattered by the virus particles; and comparing the amount of light scattered to one or more known light scattering values correlated with one or more known concentrations of the virus. The comparing step may include a standard light scattering curve including several data points, wherein the data points are based on the light scattering values and the concentrations of the virus. The method may further include the step of purifying the virus particles using a fractionation system prior to the measuring step. The fractionation system may include a chromatographic medium, such as, for example, an ion-exchange medium, a size-exclusion medium, an affinity medium, and the like. The fractionation system may include one or more components such as a chromatography column, a countercurrent distribution apparatus, a two-phase system, a gradient, or a centrifuge. The virus may be, for example, an adenovirus, human herpesvirus, human papilloma virus, adeno-associated virus, flavivirus, dengue virus, Japanese encephalitis virus, human T-cell lymphotrophic virus, hepatitis virus, human immunodeficiency virus (HIV), cytomegalovirus (CMV), Epstein-Barr virus, reovirus, vaccinia virus, parvovirus, feline leukemia virus, cauliflower mosaic virus and tomato bushy stunt virus.
In another embodiment, the invention provides a system for quantitation of virus particles including a light source adapted for directing light along a light path, a sample within the light path, a detector positioned to detect light scattered at an angle to the light path, and a recorder in communication with the detector, wherein the sample may include a quantity of particles of a virus, and wherein a portion of the light may be scattered from the path at the angle by the virus particles, and wherein the detector detects the light scattered at the angle to produce a signal that is a function of the quantity of virus particles, and wherein the signal may be communicated to the recorder and converted to a value indicating the quantity of the virus particles. The detector may be selected from the group consisting of a multi-angle detector, a dual-angle detector, and a single-angle detector. The system may further include a fractionation system in communication therewith, wherein the fractionation system receives a pre-sample including the virus particles and other components, and wherein the fractionation system separates the virus particles from the other components. The virus may be, for example, an adenovirus, human herpesvirus, human papilloma virus, adeno-associated virus, flavivirus, dengue virus, Japanese encephalitis virus, human T-cell lymphotrophic virus, hepatitis virus, human immunodeficiency virus (HIV), cytomegalovirus (CMV), Epstein-Barr virus, reovirus, vaccinia virus, parvovirus, feline leukemia virus, cauliflower mosaic virus and tomato bushy stunt virus. The quantity measured by the system of this aspect of the invention may be a concentration of virus particles per unit volume of a liquid sample. For example, such concentration may be between about 108 and 1012 virus particles/mL. Likewise, the quantity measured by the system of this aspect of the invention may be a number of virus particles in the sample. For example, such number maybe between about 108 and 1010 virus particles.